人类胚胎干细胞向造血细胞的自发分化  

Spontaneous differentiation of human embryonic stem cells into hematopoietic cells

作　　者：王建[1] 林戈[1] 赵惠萍[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《南方医科大学学报》2009年第4期602-605,610,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30300119);国家教育部博士点基金(20030533002)

摘　　要：目的研究人类胚胎干细胞自发向造血分化过程中造血干细胞和成熟血细胞标志出现时间,这将为胚胎干细胞定向诱导分化提供依据。方法将我所建立的人类胚胎干细胞系(chESC3)自发分化形成拟胚体,RT-PCR方法检测不同时间点造血相关基因KDR、Bmi1、Scl、gata2等的表达、流式细胞术检测6、8、10、12d造血干细胞标志CD34表达,并通过造血集落培养方法检测这些时间点造血集落形成能力,最后将拟胚体制备石蜡切片,免疫细胞化学检测10、12、15、18d CD45阳性阳性细胞数。结果造血干细胞早期基因KDR、Bmi1在hESCs中有表达,同时该基因随着拟胚体培养时间的延长,在第4～6天开始上调表达;造血干细胞标志性基因Scl,gata2在6～8d开始表达,并且维持高表达到12d。流式细胞术检测不同时间点hEBs中CD34阳性细胞数,发现其随时间的延长有增多的趋势,6、8、10、12d分别为(1.4±0.4)%、(3.4±1.3)%、(5.5±2.2)%、(5.1%±1.7)%;6、8、10、12d的拟胚体细胞进行集落培养检测形成的集落数在每105个拟胚体细胞中分别为0、7±2、37±11、89±29,P<0.01;集落细胞表达CD45,瑞士吉姆撒染色,可以检测到分叶核粒细胞;免疫细胞化学法对10、12、15、18d拟胚体进行切片染色,发现在这4d中可以明显观察到CD45阳性细胞的出现,并随时间的延长而数量增多,分别为:0、40.5±15.09、178.6±55.89、253.0±52.04,P<0.05。结论在自发向造血细胞分化的过程中经历了3个阶段:hESCs向胚层特异性细胞分化(前6～8d);造血干祖细胞扩增期(第8～12天);成熟血细胞大量出现期(15d以后)。Objective To characterize the time course of spontaneous differentiation of in vitro cultured human embryonic stem cells （hESCs） into hematopoietic cells to provide experimental evidence for induction of hematopoietic commitment of hESCs. Methods In human embryoid bodies （hEBs） derived from spontaneous differentiation of chESC3, a hESC cell line we established previously, the expressions of such genes as KDR, Broil, Scl and gata2 were detected by RT-PCR every other day during the 12-day differentiation to monitor the process of the hematopoiesis. The hematopoietic stem cell marker CD34 was examined using flow cytometry to evaluate the efficiency ofhematopoietic differentiation of the cells on days 6, 8, 10 and 12. The spontaneously differentiated hESCs were seeded in the hematopoietic colony culture system to study the hematopoietic colony forming ability. Immunocytochemical staining for CD45 was performed on the hEBs to examine the emergence of mature hematopoietic cells. Results The expressions of the hematopoietic stern cell-related genes KDR and Bmi-1 were detected in the hESCs, and on days 4 to 6, the two genes were upregulated with prolonged cuture of the hEBs. Sel and gata2 gene expressions were detected since 6-8 days of culture and maintained high expressions till day 12. Flow cytometry revealed a gradual increase in CD34-positive cells in the culture, with positivity rates on days 6, 8, 10, and 12 of （1.4±0.4）%, （3.4±1.3）%, （5.5±2.2）%, and （5.1± 1.7）%, respectively. The numbers of CD43-positive cell colonies on days 6, 8, 10, and 12 were 0, 7±2, 37±11, and 89±29 in each 10^5 cells, respectively. Immunocytochemical staining identified CD45-positive cells on days 10, 12, 15, and 18 in the cell colonies, with the positive cell numbers of 0, 40.5±15.09, 178.6±55.89, and 253.0±52.04, respectively. Conclusion The hEgCs undergo spontaneous hematopoietic differentiation in 3 stages, including the differentiation into germ layer-specific cells （days 6-8）, expansion period of

关 键 词：造血干细胞 胚胎干细胞 分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]